Summary: The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self-renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We have shown that Hmgb3 is a protein the binds both to transcription factors and to chromatin and used an Hmgb3 knockout mouse model to demonstrate that Hmgb3 is required to regulate the balance between differentiation and proliferation of the most primitive hematopoietic cells. We have shown that Hmgb3 regulates the expression of c-kit receptor on active HSC and are deveoping tools to demonstrate Hmgb3 binding to the kit locus. Specific Aim 1.2: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. We have identified a gene - Asridj, which is expressed in both skeletal muscle stem cells, ES cells and HSC, as well as being in several ?stem cell? databases. Retroviral transfer of the Asridj gene into bone marrow HSC inhibits hematopoietic differentiation. We are now developing knock out ES cells to further evaluate Asrij functionwill be used to identify the effects of over expression of Asridj. Project 2: We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease and the adverse events in other gene therapy trials point out the problem of inserting powerful enhancers like those from the globin locus into the genome. Specific aim 2.1: We have shown that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells, and that this leads to improved gene transfer to human hematopoietic cells in the sheep xenograft model. We are adapting the FeLV-C envelope to pseudotype lentivirus vectors to suppress globin vector instability and improve gene transfer frequency. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia without enhancer elements. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have also identified a key regulatory region in the ankyrin promoter that we will modify in an attempt to increase the activity of the ankyrin promoter to give higher levels of globin expression. We have also demonstrated a compact insulator element in the ankyrin promoter that provides protection from gene silencing in vitro and in vivo. We are currently evaluating the regulatory regions of other red cells genes to identify both enhancers and barrier elements to incorporate into our globin vectors.